Lactobacillus is a genus of bacteria in the family Lactobacteriacae, found in the intestinal tract of mammals, green plants, milk and fermenting foods. Lactic acid is produced by these bacteria via carbohydrate fermentation; thus, lactobacilli have been introduced into the manufacture of food products, such as yogurt and cheese, to enhance their quality and stability. In addition, lactobacilli form an important part of the internal microbial flora in humans and other animals. In humans, lactobacilli are present in the mouth, lower intestine and vagina.
Another type of bacteria, Eschericia coli (hereinafter, E. coli), is also present in animals and can cause serious intestinal problems such as diarrhea. The disease gastroenteritis is also caused by E. coli infection. A proper balance of the microbial populations in the intestinal tract of animals is believed to be important to provide resistance to such diseases. For a review, see W. E. Sandine et al., J. Milk Food Technol., 35, p. 691-702 (1972), incorporated by reference herein. Stress conditions can alter the balance of microbes in the intestinal tracts of humans and other animals. The basis of diseases such as gastroenteritis is thought to be a microbial imbalance in the intestinal tract. This knowledge of the importance of maintaining internal populations of "helpful" bacteria such as lactobacilli to improve health has led to attempts to administer lactobacillus to treat humans and animals. L. acidophilus has shown some effectiveness in destroying E. coli in infants suffering from diarrhea. Such therapy has included the use of strains of L. acidophilus in dried form, administered orally, for promoting L. acidophilus colonization in human intestines.
L. acidophilus has also been used in animal feed in an attempt to restore and stabilize the internal microbial balance. In some cases, animals such as pigs were found to grow better and exhibited a decreased population of E. coli when administered lactobacilli. Two patents (U.S. Pat. No. 3,343,962 and British Patent No. 1,134,206) have been issued on methods of preparation of certain lactobacilli for use in animal feed supplements.
Unfortunately, in field studies outside of the laboratory, prior preparations using known strains of Lactobacillus have proven ineffective, in part because sufficiently high numbers of viable microorganisms are not present in the preparations and the organisms have not been able to successfully colonize the subject being treated. It is thought that adhesion is a primary event in colonization by bacteria of a particular habitat, such as the intestinal tract. Bacteria are known to adhere to various surfaces, including human and animal cells. G. W. Jones, "The Attachment of Bacteria to the Surfaces of Animal Cells," in Microbial Interactions (Russig Ed.), Chapman and Hall, London (1977), incorporated by reference herein. Colonization by bacteria also appears to be important for the establishment and maintenance of both normal and disease-associated bacterial flora in humans and other animals. For example, the ability of bacteria to attach to human mucosal epithelial cells, such as vaginal cells, has been studied for possible correlation with the subsequent colonization by pathogens and bacterial invasion of underlying tissues. R. A. Mardh and L. Westrom, Infection and Immunity, 13, p. 661-666 (1976); Chan et al., Infection and Immunity, 47, p. 84-89 (1985).
Distinct differences between strains of Lactobacillus acidophilus exist in their ability to survive, initiate and maintain a population within the intestine due, in part, to differences in the ability of the various strains to adhere to the epithelial cells of different species of animals. These differences may hamper the effectiveness of the administered bacteria. Prior known strains of lactobacilli demonstrate species specificity, such that one strain of Lactobacillus acidophilus from a chicken source will not adhere to epithelia of a different species, e.g., a rat, as shown by N. Suegara et al., Infection and Immunity, 12, p. 173-179 (1975); and R. Fuller, J. Applied Bact., 45, p. 389-395 (1978). The stomach and intestinal tract also present physical challenges to the growth and survival of microorganisms, such as Lactobacillus, including the removal mechanism of the small intestine which washes out any organisms which cannot attach to the intestinal epithelial cells or multiply fast enough to avoid dilution.
It is believed that bacteria may attach to animal cells through a variety of mechanisms. In one system, cations may provide a bridge via ionic attraction between surfaces of the bacteria and epithelial cells. This system appears to be nonspecific and is calcium dependent. In another mechanism, the bacteria appear to attach by contacting receptor sites on the epithelial cells. This system is calcium independent and has been found to be species specific, suggesting that the ability to adhere (i.e., to recognize receptors) when mediated by this mechanism is under the genetic control of the species. At least one researcher has identified two subpopulations of human Lactobacillus acidophilus wherein one population requires calcium to adhere to human fetal epithelial cells. One of these strains is capable of interspecific attachment. Attachment of the other was found to be independent of calcium. This calcium independence has been found in only a few other human bacterial strains. E. G. Kleeman and T. R. Klaenhammer, J. Diary Sci., 65, p. 2063-2069 (1982). Other mechanisms of attachment may be involved. Sherman et al., Appl. Environ. Microbiol., 52, p. 302-304 (1986); Fuller et al., Am. J. Clin. Nutr., 27, p. 1305-1312 (1974).
In addition to the difficulties of isolating a Lactobacillus acidophilus strain capable of adhering to the tissue of various species of animals, difficulties in culturing the strains exist.